Primary cilia are sensory organelles involved in many physiological processes. Defects in their structure, assembly, and component sensory proteins cause a wide variety of medical disorders and developmental abnormities. Much of the fundamental knowledge about primary cilia is incomplete, due to lack of a convenient methodology to characterize the structure and assembly processes of primary cilia in their native state. In the absence of a reference three-dimensional (3D) structure of primary cilia, studies on molecular mechanisms of ciliary functions are lagging. A detailed knowledge of the assembly of wild-type cilia and their growth are necessary to characterize abnormal-growth phenotypes that are due to ciliary defects, drug stimuli or environmental changes. Without such knowledge it is difficult to determine if the recently identified regulatory reagents of ciliogeness can restore or maintain normal cilium structure and if they can be used for potential drug development. The objectives of this proposal are to produce the first detailed 3D structure of native (vitreously frozen) primary cilia using cryo-electron tomography (cryo-ET), and to determine the growth behavior of primary cilia by correlative light and electron microscopy. Our ground-breaking method development has provided us the first- ever electron micrographs and tomograms of vitreously frozen primary cilia of kidney cells. Unexpectedly, our study also revealed small vesicles immediately beneath the ciliary membrane. This important finding will allow us to test the hypothesis that the assembly of primary cilia involves intraflagellar transpor (IFT) of membrane vesicles. We will carry out our research through the following two specific aims: (1) We will determine the 3D structure of the wild-type kidney primary cilium by cryo-ET; (2) We will study the growth behavior of primary cilia, and will determine whether growth is related to IFT of intra-ciliary vesicles. The proposed research is highly significant because it wil not only provide the first detailed native structure of primary cilia, but it will also establish teir growth behavior in kidney cells. We will test an important hypothesis about the functional roles of intra-ciliary vesicles that would potentially introduce a paradigm shift in our understanding about how membrane components target to the cilium. Our study will be the first step in a continuum of research to systematically characterize primary cilia in order to understand the roles of various ciliary proteins or their complexes. Furthermore, the methods developed during the course of the proposed study will provide a new platform for future research on primary cilia. This platform can be used, for example, to study primary cells from ciliopathological animal models or from biopsy specimens of patients with cystic diseases, and it will contribute to in vitr drug screening and therapy development.